To achieve their diverse range of biological functions, RNA molecules must fold into specific tertiary structures that create catalytic centers or ligand recognition sites. Despite the recent emergence of a wealth of information on the three-dimensional structures of RNA molecules and RNA-protein complexes, culminating in structures of ribosomal particles, the mechanisms by which RNA adopts these structures are poorly understood. The broad, long-term objective of this proposal is to understand the mechanism of RNA folding. Fundamental features of RNA folding include conformational search, metal ion binding and productive intermediates. Additionally, most large RNA molecules require protein cofactors in order to fold correctly and efficiently. Hence, we will continue to analyze tertiary structure formation in the hairpin ribozyme, an autonomously folding RNA, and we will also undertake new studies of the folding of the 7SL RNA from the mammalian signal recognition particle (SRP), which folds in the presence of multiple protein chaperones. By studying both systems, we hope to learn about the general principles of RNA folding. Moreover, the results will facilitate the design of improved ribozymes for therapeutic applications in gene therapy and, eventually, may lead to new cancer therapies based on arrest of protein synthesis by the SRP machinery. The specific aims are: (1) Elucidate the role of loop rearrangements and dynamic loop structure in tertiary structure formation in the hairpin ribozyme. (2) Test the proposed conformational search model for tertiary structure formation in the hairpin ribozyme and identify determinants of the search. (3) Monitor RNA folding transitions during assembly of the Alu domain of the signal recognition particle. (4) Elucidate the mechanism of the initial assembly of the S-domain of the signal recognition particle and characterize associated RNA folding transitions. To address these goals, we will apply a range of ensemble and single-molecule fluorescence methods to dissect the RNA folding pathways and to monitor dynamic conformational transitions during folding. Additionally, the experimental methods developed during this project will provide new tools for studying the conformational dynamics and folding of RNA and will be broadly applicable.